G protein-coupled receptors mediate extracellular signals from, for example, hormones, neurotransmitters, light and odorants via G proteins into the interior of the cell, and various effects can be initiated via an intracellular signal cascade. G proteins normally consist of three different subunits (alpha, beta, and gamma). Various heterodimeric G proteins which differ in receptor specificity and effect are known. The G proteins are activated by GTP. A well-known G protein is transducin from the vision process.
G protein-coupled receptors (GPCR) play an important part in a large number of physiological processes. They are one of the largest protein families known. It is currently estimated that about 1,000 genes in the human genome code for this class of receptors. GPCR are membrane proteins with 7 transmembrane α-helices. A large number of medicaments displays its effect via GPCRs.
GPCRs are involved especially in signal processing and control of the organism and therefore play a important part in maintaining the function of the intact organism.
The binding of an extracellular ligand leads to a conformational change in the relevant GPCR. The conformational change creates the preconditions for interaction with the respectively associated G protein. The G protein in turn initiates an intracellular signal cascade which is characteristic of the relevant cell type. The so-called second messengers are characteristic of intracellular signal cascades. By these are meant low molecular weight compounds such as, for example, cAMP (cyclic adenosine monophosphate), cGMP (cyclic guanosine monophosphate) or Ca2+. The intracellular signaling is controlled by changes in the concentration of the second messengers. The G proteins and their subunits interact for this purpose with proteins such as adenylate cyclase, phospholipase C or ion channels. The change in the concentration of the second messenger in turn brings about an activation or inactivation of other proteins, especially of kinases and phosphatases. The signal finally terminates in a response typical of the particular cell assembly, for example the expression of a protein.
The heterotrimeric G proteins are located on the inside of the plasma membrane. An activated receptor makes contact with the G protein heterotrimer, which then dissociates an α subunit and the βγ complex. Both the activated α subunit and the βγ complex are able to influence intracellular effector proteins. The G protein α subunit family can be divided into various classes. Known examples are the Gαs, Gαi, Gαq and Gα12 classes. GPCRs are classified according to the activated G proteins.
GPCRs of the Gs class mediate, via activation of Gαs, the stimulation of adenylate cyclase and increase the intracellular cAMP concentration. GPCRs of the Gi class bring about, via activation of GαI, an inhibition of adenylate cyclase and reduce the intracellular cAMP. GPCRs of the Gq class in turn achieve, via activation of Gαq, a stimulation of various PLCβ isoforms and lead, via hydrolysis of membrane-bound phosphatidylinositol 4,5-biphosphate, to diacylglycerol and inositol triphosphate (IP3). IP3 releases Ca2+ from intracellular stores. Most GPCRs are able to make contact with only one G protein β subunit family, i.e. they have selectivity for a particular signal transduction pathway.
G proteins with altered receptor specificity and different attachment to a signal transduction pathway can be constructed by joining together components from different G proteins to give hybrid G proteins by the methods of molecular biology and biochemistry.
Hybrid G proteins are fusion constructs which combine within one protein sequences of different Gα subunits. Thus, for example, it is possible by fusing the receptor recognition region of Gαi with the effector activation region of Gαq to produce a Gαq/i hybrid which receives the signals of Gi-coupled receptors but switches on the Gαq PLCβ signal transduction pathway. Such a hybrid in which the C-terminal 5 amino acids of Gαq have been replaced by the corresponding Gαi sequence (Gαiq5) was described for the first time by Conklin et al. Nature 363, 274-276 (1993).
This “rerouting” of receptors has the advantage that the assay endpoint (increase in intracellular Ca2+ concentration compared with inhibition of adenylate cyclase) is more easily accessible by measurement techniques and can be used in high throughput screening.
The nucleotide sequence and amino acid sequence of the GPR45 like/GPR63 receptor are known (Genbank: NM—030784; TREMBL:Q9b2i6). The nucleotide sequence of the GPR45 like/GPR63 receptor is set forth in SEQ ID NO:1, and its amino acid sequence in SEQ ID NO:2.
Prior to the inventor's discovery, no one knew which ligands bound to this receptor, making it impossible to identify any agonists or antagonists to the receptor using current laboratory methods. Agonists and antagonists are usually defined organic molecules with a precise structure and a reproducible process of preparation. They are an important research tool: only with the aid of such compounds is it possible to investigate the function of this receptor in various stages of development, in different tissues, including in normal and pathologically altered tissues, and in environments subject to different external influences.